H. A. Frohman1,2, P. G. Rychahou1,2, D. S. Watt3,4, Y. Y. Zaytseva2,5, C. Liu2,3, N. Roller2, K. Wang1, B. M. Evers1,2 1University Of Kentucky,Department Of Surgery,Lexington, KY, USA 2University Of Kentucky,Markey Cancer Center,Lexington, KY, USA 3University Of Kentucky,Department Of Molecular And Cellular Biochemistry,Lexington, KY, USA 4University Of Kentucky,Center For Molecular Medicine, Organic Synthesis Core,Lexington, KY, USA 5University Of Kentucky,Department Of Toxicology And Cancer Biology,Lexington, KY, USA
Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US with the majority of deaths due to metastatic disease. Current chemotherapeutic regimens involve highly toxic agents, which limits their utility; therefore, more effective and less toxic agents are required to see a reduction in CRC morbidity and mortality. Novel fluorinated N,N’-diarylureas (FND) were developed and characterized by our group as potent activators of adenosine monophosphate-activated kinase (AMPK) that inhibit cell cycle progression. The purpose of this study was to determine the effect of a lead FND compound, FND4b, either alone or combined with PI103 (a PI3K inhibitor) or SN38 (active metabolite of irinotecan) on cell cycle arrest and apoptosis of established CRC cell lines and primary CRC lines established from patient-derived xenografts (PDXs).
Methods: We tested the effects of FND4b, PI103 and SN38 on cell cycle arrest and apoptosis using commercially available CRC cell lines (HT29 and HCT116) and primary cell lines from PDXs established from patients following surgical resection. Briefly, CRC tissues were implanted into NOD scid gamma (NSG) and PDXs (Pt93, Pt130, Pt2377-Primary Tumor [PT], Pt2377-Liver Metastasis [LM]) were established after sequential generations in NSG mice. These PDX models were authenticated as unique human cell lines and genetic profile of 198 oncogenes was determined by Next Generation Sequencing.
Results: Treatment with FND4b for 24h resulted in a marked induction of phosphorylated AMPK expression and a concomitant reduction in Cyclin D1 expression in all six CRC cell lines. Cleaved PARP expression was also notably increased in CRC cells treated with FND4b, which indicated increased apoptosis. Three of the six CRC cell lines had PI3K mutations (HT29, Pt2377-PT, Pt2377-LM), as determined by oncogenic mutation profiling. Other mutations included KRAS, APC, and BRAF. Regardless of the genetic profile of the CRC cells, FND4b treatment resulted in decreased cell proliferation. When CRC cells were treated with combinations of FND4b, PI103, and SN38, there was no change in cell cycle arrest or apoptosis, as compared to treatment with FND4b alone.
Conclusion: Our findings identify FND4b as a novel and effective inhibitor of CRC growth either alone or in combination with PI103 and SN38. Moreover, FND4b activates AMPK at micromolar concentrations, which consistently result in cell cycle arrest and apoptosis in commercially available and PDX-derived CRC cells. Future studies will delineate the effectiveness of FND4b in an in vivo CRC model.